The invention relates to proteins which are upregulated in injured or regenerating tissues, as well as to the DNA encoding these proteins. The invention further relates to therapeutic compositions and methods of treatment encompassing these proteins.
A dynamic remodeling of tissue architecture occurs during development and during tissue repair after injury. Herein are presented results of studies of this process, using a model of kidney injury caused by an ischemia-reperfusion insult.
The kidney is able to repair damage to the proximal tubule epithelium through a complex series of events involving cell death, proliferation of surviving proximal tubule epithelial cells, formation of poorly differentiated regenerative epithelium over the denuded basement membrane, and differentiation of the regenerative epithelium to form fully functional proximal tubule epithelial cells (Wallin et al., Lab. Invest. 66:474-484, 1992; Witzgall et al., Mol. Cell. Biol. 13:1933-1942, 1994; Ichimura et al., Am. J. Physiol. 269: F653-662, 1995; Thadhani et al., N. Engl. J. Med. 334:1448-1460, 1996). Growth factors such as IGF, EGF, and HGF have been implicated in this process of repair, as has the endothelial cell adhesion molecule ICAM-1. However, the mechanisms by which the tubular epithelial cells are restored are still not understood.
To identify molecules involved in processes of injury and repair of the tubular epithelium, differences were analyzed in the mRNA populations between injured/regenerating and normal kidneys using representational difference analysis (RDA). RDA is a PCR-based method for subtraction which yields target tissue or cell specific cDNA fragments by repetitive subtraction and amplification (Hubank and Schutz, Nucl. Acids Res. 22:5640-5648, 1994).
The invention generally provides Kidney Injury-related Molecules (each of which is henceforth called a xe2x80x9cKIMxe2x80x9d) which are upregulated in renal tissue after injury to the kidney. The KIM proteins and peptides of the invention, as well as their agonists and antagonists, and their corresponding nucleic acids are useful in a variety of therapeutic interventions.
The invention provides a purified and isolated DNA molecule having a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. The invention also includes the complementary strands of these sequences, DNA molecules which hybridize under stringent conditions to the aforementioned DNA molecules, and DNA molecules which, but for the degeneracy of the genetic code, would hybridize to any of the DNA molecules defined above. These DNA molecules may be recombinant, and may be operably linked to an expression control sequence.
The invention further provides a vector comprising a purified and isolated DNA molecule having a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or one of the other DNA molecules defined above. This vector may be a biologically functional plasmid or viral DNA vector. One embodiment of the invention provides a prokaryotic or eukaryotic host cell stably transformed or transfected by a vector comprising a DNA molecule of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In another embodiment of the invention, a process is provided for the production of a KIM polypeptide product encoded by a DNA molecule as described above; the process involves growing, under suitable culture conditions, prokaryotic or eukaryotic host cells transformed or transfected with the DNA molecule in a manner allowing expression of the DNA molecule, and recovering the polypeptide product of said expression.
A purified and isolated human KIM protein substantially free of other human proteins is specifically within the invention, as is a process for the production of a polypeptide product having part or all of the primary structural conformation and the biological activity of a KIM protein. KIM proteins of the invention may have an amino acid sequence which comprises SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or may be, a variant of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, or a purified and isolated protein encoded by the DNA of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. These proteins can be provided substantially free of other human proteins. The invention further includes variants of these proteins, such as soluble variants or fusion proteins. KIM fusion proteins of the invention may comprise an immunoglobulin, a toxin, an imageable compound or a radionuclide.
The invention also provides a specific antibody, such as a monoclonal antibody (MAb), to the KIM proteins described above. The present antibody binds to any epitope that is unique to a KIM protein disclosed herein. In some embodiments, the epitope is displayed on the surface of a cell expressing KIM. Antigen-binding fragments of the antibody are also provided herein, preferably Fab, Fab2, Fabxe2x80x2 and Fv fragments, whether produced by chemical or enzymatic cleavage, or by molecular engineering techniques. Engineered versions of the present antibody include chimeric, humanized and human antibodies, and antibody fusion proteins. Monoclonal antibodies (MAbs) of the present invention can be of mouse, rat, hamster or human origin. An exemplary MAb of the invention is the murine AKG7 MAb disclosed herein, which binds specifically to the human KIM polypeptide disclosed herein. Anti-KIM antibodies of the present invention may be conjugated or fused to a therapeutic agent, toxin, imageable compound or radionuclide. Exemplary therapeutic agents include cytokines, lymphokines, trophic factors, survival factors, chemokines and chemoattractants. Exemplary toxins include ricin and diphtheria toxin. Exemplary imageable compounds include luminescent proteins (e.g., luciferin), fluorescent proteins (e.g., green fluorescent protein), haptens (e.g., biotin), and radioactively labeled proteins. Exemplary radionuclides include any radionuclide used for medical imaging purposes. The invention further encompasses all hybridoma cell lines and engineered host cells which produce antibodies of the invention.
Pharmaceutical compositions are also within the scope of the invention. A pharmaceutical composition of the invention may comprise a therapeutically effective amount of a KIM protein or anti-KIM antibody of the invention, along with a pharmacologically acceptable carrier.
Diagnostic methods are within the invention, such as assessing the presence or course of resolution of renal injury by measuring the concentration of KIM in urine, serum, or urine sediment of patients who have or who are at risk of developing renal disease. Other diagnostic methods that are within the invention include assessing KIM expression level in kidney tissue (e.g., in kidney biopsy tissue) of patients who have, are suspected of having, or are at risk of developing renal cancer (e.g., renal carcinoma). The present methods involve contacting an appropriate tissue or fluid sample derived from the patient being diagnosed, with a KIM antibody or a KIM probe (as the case may be), under binding conditions. Complexes formed by the binding of antibody or probe to KIM protein or nucleic acid (e.g., RNA) in the sample are detected by standard techniques. The presence or abnormal elevation of KIM protein in urine or serum is expected to correlate with renal failure or renal disease. The presence or abnormal elevation of KIM gene expression in renal cells or tissue is expected to correlate with disease processes, particularly carcinogenesis. Such correlations are expected to be useful in the prognostication, staging and clinical management of diseases or other conditions deleterious to renal tissue and/or renal function.
Methods of treatment of the invention include treating patients with therapeutically; effective amounts of KIM, KIM variants, KIM analogs, KIM fusion proteins, KIM agonists, and antibodies to KIM or to KIM ligands. Other therapeutic compounds of the invention include KIM ligands, anti-KIM antibodies, and fusions proteins of KIM ligands. These compounds can be useful in therapeutic methods which either stimulate or inhibit cellular responses that are dependent on KIM function.
Further methods of the invention inhibit the growth of KIM-expressing tumor cells by contacting the cells with a fusion protein of a KIM ligand and either a toxin or radionuclide, or with an anti-KIM antibody conjugated to a toxin or to a radionuclide. Likewise, growth of tumor cells which express KIM ligand may be inhibited by contacting the cells with a fusion protein of a KIM and either a toxin or radionuclide, or with an anti-KIM ligand antibody conjugated to a toxin or to a radionuclide.
The invention also encompasses methods of gene therapy. These include a method of treating a subject with a renal disorder, a method of promoting growth of new tissue in a subject, and a method of promoting survival of damaged tissue in a subject, wherein each method comprises administering to the subject a vector which includes DNA comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
The compounds of the invention are also useful for imaging tissues, either in vitro or in vivo. One such method involves targeting an imageable compound to a cell expressing a protein of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, comprising contacting the cell with either a monoclonal antibody of the invention or a fusion protein comprising a protein as described above, fused to an imageable compound. For in vivo methods, the cell is within a subject, and the protein or the monoclonal antibody is administered to the subject.
The invention also includes diagnostic methods, such as a method of identifying damage or regeneration of renal cells in a subject, comprising comparing the level of expression of either SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 in renal cells of the subject to a control level of expression of the sequence in control renal cells. Another method of the invention includes identifying upregulation of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 in cells comprising contacting the cells with an antisense probe and measuring hybridization to RNA within the cell.
A further embodiment of the diagnostic methods of the invention includes assessing the presence or concentration of a molecule of the invention either in urine, serum, or other body fluids, or in urine sediment or tissue samples. The measured injury-related molecule can be correlated with the presence, extent or course of a pathologic process. This correlation can also be used to assess the efficacy of a therapeutic regime.